Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site.

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo.

Conformational changes in the catalytic region are responsible for heat-induced activation of hyperthermophilic homoserine dehydrogenase.

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.

Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation.

Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (ß/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-ß-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (ß/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (ß/α)8-barrel domain.

3D-QSAR Studies of 1,2,4-Oxadiazole Derivatives as Sortase A Inhibitors.

Sortase A (SrtA) is an enzyme that catalyzes the attachment of proteins to the cell wall of Gram-positive bacterial membrane, preventing the spread of pathogenic bacterial strains. Here, one class of oxadiazole compounds was distinguished as an efficient inhibitor of SrtA via the "S. aureus Sortase A" substrate-based virtual screening. The current study on 3D-QSAR was done by utilizing preparation of the structure in the Schrödinger software suite and an assessment of 120 derivatives with the crystal structure of 1,2,4-oxadiazole which was extracted from the PDB data bank. The docking operation of the best compound in terms of pMIC (pMIC = 2.77) was done to determine the drug likeliness and binding form of 1,2,4-oxadiazole derivatives as antibiotics in the active site. Using the kNN-MFA way, seven models of 3D-QSAR were created and amongst them, and one model was selected as the best. The chosen model based on q 2 (pred_r 2) and R 2 values related to the sixth factor of PLS illustrates better and more acceptable external and internal predictions. Values of crossvalidation (pred_r 2), validation (q 2), and F were observed 0.5479, 0.6319, and 179.0, respectively, for a test group including 24 molecules and the training group including 96 molecules. The external reliability outcomes showed that the acceptable and the selective 3D-QSAR model had a high predictive potential (R 2 = 0.9235) which was confirmed by the Y-randomization test. Besides, the model applicability domain was described successfully to validate the estimation of the model.

Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα.

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

Isoliquiritigenin, a potent human monoamine oxidase inhibitor, modulates dopamine D1, D3, and vasopressin V1A receptors.

Isoliquiritigenin (= 4,2',4'-Trihydroxychalcone) (ILG) is a major constituent of the Glycyrrhizae Rhizoma that has significant neuroprotective functions. In the present study, we re-examined the potential of ILG to inhibit human monoamine oxidase (hMAO) in vitro and established its mechanism of inhibition through a kinetics study and molecular docking examination. ILG showed competitive inhibition of hMAO-A and mixed inhibition of hMAO-B with IC50 values of 0.68 and 0.33 µM, respectively, which varied slightly from the reported IC50 values. Since ILG has been reported to reduce dopaminergic neurodegeneration and psychostimulant-induced toxicity (both of which are related to dopamine and vasopressin receptors), we investigated the binding affinity and modulatory functions of ILG on dopamine and vasopressin receptors. ILG was explored as an antagonist of the D1 receptor and an agonist of the D3 and V1A receptors with good potency. An in silico docking investigation revealed that ILG can interact with active site residues at target receptors with low binding energies. These activities of ILG on hMAO and brain receptors suggest the potential role of the compound to ameliorate dopaminergic deficits, depression, anxiety, and associated symptoms in Parkinson's disease and other neuronal disorders.

Experimental and in silico studies of competitive inhibition of family GH10 Aspergillus fumigatus xylanase A by Oryza sativa xylanase inhibitor protein.

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn2+ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (Ki) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was -30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239OsXIP stably interacted with the catalytic site E140AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104AfXylA10/V99AfXylA10-Q5OsXIP, R256AfXylA10-E235OsXIP, D155AfXylA10-Y243OsXIP and D145AfXylA10-R194OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP.

The Dynamic Network of RNP RNase P Subunits.

Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5' end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies.

Conserved Trigger Loop Histidine of RNA Polymerase II Functions as a Positional Catalyst Primarily through Steric Effects.

In all domains of life, multisubunit RNA polymerases (RNAPs) catalyze both the extension of mRNA transcripts by nucleotide addition and the hydrolysis of RNA, which enables proofreading by removal of misincorporated nucleotides. A highly conserved catalytic module within RNAPs called the trigger loop (TL) functions as the key controller of these activities. The TL is proposed to act as a positional catalyst of phosphoryl transfer and transcript cleavage via electrostatic and steric contacts with substrates in its folded helical form. The function of a near-universally conserved TL histidine that contacts NTP phosphates is of particular interest. Despite its exceptional conservation, substitutions of the TL His with Gln support efficient catalysis in bacterial and yeast RNAPs. Unlike bacterial TLs, which contain a nearby Arg, the TL His is the only acid-base catalyst candidate in the eukaryotic RNAPII TL. Nonetheless, replacement of the TL His with Leu is reported to support cell growth in yeast, suggesting that even hydrogen bonding and polarity at this position may be dispensable for efficient catalysis by RNAPII. To test how a TL His-to-Leu substitution affects the enzymatic functions of RNAPII, we compared its rates of nucleotide addition, pyrophosphorolysis, and RNA hydrolysis to those of the wild-type RNAPII enzyme. The His-to-Leu substitution slightly reduced rates of phosphoryl transfer with little if any effect on intrinsic transcript cleavage. These findings indicate that the highly conserved TL His is neither an obligate acid-base catalyst nor a polar contact for NTP phosphates but instead functions as a positional catalyst mainly through steric effects.

Structural Insights in Mammalian Sialyltransferases and Fucosyltransferases: We Have Come a Long Way, but It Is Still a Long Way Down.

Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer.

Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases.

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme-reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

Structural basis of the complementary activity of two ketosynthases in aryl polyene biosynthesis.

Aryl polyenes (APE) are one of the most widespread secondary metabolites among gram-negative bacteria. In Acinetobacter baumannii, strains belonging to the virulent global clone 2 (GC2) mostly contain APE biosynthesis genes; its relevance in elevated pathogenicity is of great interest. APE biosynthesis gene clusters harbor two ketosynthases (KSs): the heterodimeric KS-chain length factor complex, ApeO-ApeC, and the homodimeric ketoacyl-acyl carrier protein synthase I (FabB)-like KS, ApeR. The role of the two KSs in APE biosynthesis is unclear. We determined the crystal structures of the two KSs from a pathogenic A. baumannii strain. ApeO-ApeC and ApeR have similar cavity volumes; however, ApeR has a narrow cavity near the entrance. In vitro assay based on the absorption characteristics of polyene species indicated the generation of fully elongated polyene with only ApeO-ApeC, probably because of the funnel shaped active site cavity. However, adding ApeR to the reaction increases the throughput of APE biosynthesis. Mutagenesis at Tyr135 in the active site cavity of ApeR reduces the activity significantly, which suggests that the stacking of the aryl group between Tyr135 and Phe202 is important for substrate recognition. Therefore, the two KSs function complementarily in the generation of APE to enhance its production.

The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect.

Aerobic glycolysis in cancer cells, also known as the 'Warburg effect', is driven by hyperactivity of lactate dehydrogenase A (LDHA). LDHA is thought to be a substrate-regulated enzyme, but it is unclear whether a dedicated intracellular protein also regulates its activity. Here, we identify the human tumor suppressor folliculin (FLCN) as a binding partner and uncompetitive inhibitor of LDHA. A flexible loop within the amino terminus of FLCN controls movement of the LDHA active-site loop, tightly regulating its enzyme activity and, consequently, metabolic homeostasis in normal cells. Cancer cells that experience the Warburg effect show FLCN dissociation from LDHA. Treatment of these cells with a decapeptide derived from the FLCN loop region causes cell death. Our data suggest that the glycolytic shift of cancer cells is the result of FLCN inactivation or dissociation from LDHA. Together, FLCN-mediated inhibition of LDHA provides a new paradigm for the regulation of glycolysis.

The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme.

Among industrially important pyridoxal-5'-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

Insight into the function of active site residues in the catalytic mechanism of human ferrochelatase.

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution.

Carboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. Maintenance of carboxysome distribution protein A (McdA), a partition protein A (ParA)-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to the carboxysome-localized Maintenance of carboxysome distribution protein B (McdB). As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. How the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP, and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos.

Molecular Dynamics Simulations of a Cytochrome P450 from Tepidiphilus thermophilus (P450-TT) Reveal How Its Substrate-Binding Channel Opens.

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl's side chain tightens and relaxes the opening to the channel in conjunction with the arginyl's, though the latter's side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.